Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels

Abstract

The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca 2+ -calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca 2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.

1. An isolated nucleic acid encoding a modified mammalian olfactory cyclic nucleotide-gated ion channel alpha subunit, wherein said channel comprises a mutation, wherein said mutation comprises a substitution at a residue corresponding to position 583 of SEQ ID NO:8, and wherein said channel has increased cAMP sensitivity, and decreased cGMP sensitivity as compared to a wild type channel. 
     
     
       2. The nucleic acid of  claim 1 , wherein said substitution at a residue corresponding to position 583 of SEQ ID NO:8 is selected from the group consisting of E583M, E583V, E583L and E583I. 
     
     
       3. The nucleic acid of  claim 1 , wherein said cGMP-induced current is 40% or less than said cAMP-induced current. 
     
     
       4. The nucleic acid of  claim 1 , wherein said increased cAMP sensitivity comprises a K 1/2  at least ten-fold lower than that observed for a nucleic acid encoding a wild type channel. 
     
     
       5. The nucleic acid of  claim 1 , wherein said decreased cGMP sensitivity comprises a K 1/2  at least ten-fold higher than that observed for a nucleic acid encoding a wild type channel. 
     
     
       6. The nucleic acid of  claim 1 , wherein said mutation further comprises a substitution at a residue corresponding to position 460 of SEQ ID NO:8. 
     
     
       7. The nucleic acid of  claim 6 , wherein said substitution at a residue corresponding to position 460 of SEQ ID NO:8 is selected from the group consisting of C460W, C460F and C460Y. 
     
     
       8. The nucleic acid of  claim 6 , wherein said mutation further comprises a 61–90 deletion at residues corresponding to positions 61–90 of SEQ ID NO:8. 
     
     
       9. An isolated nucleic acid encoding a modified rat olfactory cyclic nucleotide-gated ion channel alpha subunit, wherein said channel alpha subunit comprises a glutamic acid (E) to methionine (M) substitution at a residue corresponding to position 583 of SEQ ID NO:8. 
     
     
       10. The isolated nucleic acid of  claim 9 , wherein said channel alpha subunit further comprises a cysteine (C) to tryptophan (W) substitution at a residue corresponding to position 460 of SEQ ID NO:8. 
     
     
       11. The isolated nucleic acid of  claim 10 , wherein said channel alpha subunit further comprises a 61–90 deletion at residues corresponding to positions 61–90 of SEQ ID NO:8. 
     
     
       12. The isolated nucleic acid of  claim 9 , wherein said channel comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. 
     
     
       13. An expression vector comprising the nucleic acid of  claim 9 . 
     
     
       14. The expression vector of  claim 13 , wherein said vector is a recombinant adenovirus vector. 
     
     
       15. An isolated host cell comprising the expression vector of  claim 13 , wherein said host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. 
     
     
       16. The host cell of  claim 15 , wherein said eukaryotic cell is selected from the group consisting of a human embryonic kidney-293 cell and a rat GH4C1 pituitary cell. 
     
     
       17. An isolated polypeptide of a modified mammalian olfactory cyclic nucleotide-gated ion channel alpha subunit, wherein said channel comprises a mutation, wherein said mutation comprises a substitution at a residue corresponding to position 583 of SEQ ID NO:8, and wherein said channel has increased cAMP sensitivity, and decreased cGMP sensitivity as compared to a wild type channel.